Method of monitoring haptotaxis

ABSTRACT

The present invention discloses a method of monitoring haptotaxis comprising: providing a device for monitoring haptotaxis having a housing defining a chamber. The chamber includes: a first well region including at least one first well, the first well region configured to receive a test agent therein and further including biomolecules immobilized therein; a second well region including at least one second well, the second well region configured to receive a sample comprising cells therein and further being horizontally offset with respect to the first well region in a test orientation of the device; and a channel region with biomolecules immobilized therein and including at least one channel connecting the first well region and the second well region with one another. The method also includes forming a surface concentration gradient along a longitudinal axis of the chamber by decreasing the concentration of biomolecules from the at least one first well to the at least one second well; placing a first sample comprising cells in the at least one second well; and monitoring haptotaxis of the cells.

RELATED APPLICATIONS

[0001] This application claims the benefit of and incorporates herein byreference, in their entirety: U.S. application Ser. No. 09/709,776,filed on Nov. 8, 2000; U.S. Provisional Application No. 60/307,886,filed on Jul. 27, 2001; U.S. Provisional Application No. 60/312,405,filed on Aug. 15, 2001; U.S. Provisional Application No. 60/323,742,filed on Sep. 21, 2001; U.S. Provisional Application No. 60/328,103,filed on Oct. 11, 2001; U.S. Provisional Application No. 60/330,456,filed on Oct. 22, 2001; U.S. Provisional Application No. ______, filedon Mar. 12, 2002 and entitled, “Cell Motility and Chemotaxis Test Deviceand Method of Using Same.”

FIELD OF THE INVENTION

[0002] The present invention relates generally to a method of monitoringhaptotaxis.

BACKGROUND

[0003] Test devices, such as those used in chemotaxis, haptotaxis andchemoinvasion are well known. Such devices are disclosed for example inU.S. Pat. Nos. 6,329,164, 6,238,874, and 5,302,515.

[0004] Three processes involved in cell migration are chemotaxis,haptotaxis, and chemoinvasion. Chemotaxis is defined as the movement ofcells induced by a concentration gradient of a soluble chemotacticstimulus. Haptotaxis is defined as the movement of cells in response toa concentration gradient of a substrate-bound stimulus. Chemoinvasion isdefined as the movement of cells into and/or through a barrier or gelmatrix. The study of chemotaxis/haptotaxis and chemoinvasion and theeffects of external stimuli on such behavior are prevalent throughoutcontemporary biological research. Generally, this research involvesexposing a cell to external stimuli and studying the cell's reaction. Byplacing a living cell into various environments and exposing it todifferent external stimuli, both the internal workings of the cell andthe effects of the external stimuli on the cell can be measured,recorded, and better understood.

[0005] A cell's migration in response to a chemical stimulus is aparticularly important consideration for understanding various diseaseprocesses and accordingly developing and evaluating therapeuticcandidates for these diseases. By documenting the cell migration of acell or a group of cells in response to a chemical stimulus, such as atherapeutic agent, the effectiveness of the chemical stimulus can bebetter understood. Typically, studies of disease processes in variousmedical fields, such as oncology, immunology, angiogenesis, woundhealing, and neurobiology involve analyzing the chemotactic and invasiveproperties of living cells. For example, in the field of oncology, cellmigration is an important consideration in understanding the process ofmetastasis. During metastasis, cancer cells of a typical solid tumormust loosen their adhesion to neighboring cells, escape from the tissueof origin, invade other tissues by degrading the tissues' extracellularmatrix until reaching a blood or lymphatic vessel, cross the basallamina and endothelial lining of the vessel to enter circulation, exitfrom circulation elsewhere in the body, and survive and proliferate inthe new environment in which they ultimately reside. Therefore, studyingthe cancer cells' migration may aid in understanding the process ofmetastasis and developing therapeutic agents that potentially inhibitthis process. In the inflammatory disease field, cell migration is alsoan important consideration in understanding the inflammatory response.During the inflammation response, leukocytes migrate to the damagedtissue area and assist in fighting the infection or healing the wound.The leukocytes migrate through the capillary adhering to the endothelialcells lining the capillary. The leukocytes then squeeze between theendothelial cells and use digestive enzymes to crawl across the basallamina. Therefore, studying the leukocytes migrating across theendothelial cells and invading the basal lamina may aid in understandingthe inflammation process and developing therapeutic agents that inhibitthis process in inflammatory diseases such as adult respiratory distresssydrome (ARDS), rheumotoid arthritis, and inflammatory skin diseases.Cell migration is also an important consideration in the field ofangiogenesis. When a capillary sprouts from an existing small vessel, anendothelial cell initially extends from the wall of the existing smallvessel generating a new capillary branch and pseudopodia guide thegrowth of the capillary sprout into the surrounding connective tissue.New growth of these capillaries enables cancerous growths to enlarge andspread and contributes, for example, to the blindness that can accompanydiabetes. Conversely, lack of capillary production can contribute totissue death in cardiac muscle after, for example, a heart attack.Therefore studying the migration of endothelial cells as new capillariesform from existing capillaries may aid in understanding angiogenesis andoptimizing drugs that block vessel growth or improve vessel function. Inaddition, studying cell migration can also provide insight into theprocesses of tissue regeneration, organ transplantation, autoimmunediseases, and many other degenerative diseases and conditions.

[0006] Cell migration assays are often used in conducting these types ofresearch. Commercially available devices for creating such assays aresometimes based on or employ a transwell system (a vessel partitioned bya thin porous membrane to form an upper compartment and a lowercompartment). To study cell chemotaxis, cells are placed in the uppercompartment and a migratory stimulus is placed in the lower compartment.After a sufficient incubation period, the cells are fixed, stained, andcounted to study the effects of the stimulus on cell chemotaxis acrossthe membrane.

[0007] To study chemoinvasion, a uniform layer of a MATRIGEL™ matrix isplaced over the membrane to occlude the pores of the membrane. Cells areseeded onto the MATRIGEL™ matrix in the upper compartment and achemoattractant is placed in the lower compartment. Invasive cellsattach to and invade the matrix passing through the porous membrane.Non-invasive cells do not migrate through the occluded pores. After asufficient incubation period, the cells may be fixed, stained, andcounted to study the effects of the stimulus on cell invasion across themembrane.

[0008] The use of transwells has several shortcomings. Assays employingtranswells require a labor-intensive protocol that is not readilyadaptable to high-throughput screening and processing. Because of theconfiguration of a transwell system, it is difficult to integrate withexisting robotic liquid handling systems and automatic image acquisitionsystems. Therefore, much of the screening and processing, such ascounting cells, is done manually which is a time-consuming, tedious, andexpensive process. Cell counting is also subjective and often involvesstatistical approximations. Specifically, due to the time and expenseassociated with examining an entire filter, only randomly selectedrepresentative areas may be counted. Moreover, even when these areas arecounted, a technician must exercise his or her judgment when accountingfor a cell that has only partially migrated through the filter.

[0009] Transwell-based assays have intrinsic limitations imposed by thethin membranes utilized in transwell systems. The membrane is only 50-30microns (μm) thick, and a chemical concentration gradient that formsacross the membrane is transient and lasts for a short period. If a cellchemotaxis assay requires the chemotactic gradient to be generated overa long distance (>100-200 μm) and to be stable over at least two hours,currently available transwell assays cannot be satisfactorily performed.

[0010] Notwithstanding the above, perhaps the most significantdisadvantage of transwells is the lack of real-time observation ofchemotaxis and chemoinvasion. In particular, the changes in cellmorphology during chemotaxis cannot be observed in real-time with theuse of transwells. In transwells, when the cells are fixed to a slide,as required for observation, they are killed. Consequently, once a cellis observed it can no longer be reintroduced into the assay or studiedat subsequent periods of exposure to a test agent. Therefore, in orderto study the progress of a cell and the changes in a cell's morphologyin response to a test agent, it is necessary to run concurrent samplesthat may be slated for observation at various time periods before andafter the introduction of the test agent. In light of the multiplesamples required for each test, in addition to the positive and negativecontrols required to obtain reliable data, a single chemotaxis assay canrequire dozens of filters, each of which needs to be individuallyexamined and counted-an onerous and time-consuming task.

[0011] More recently, devices for measuring chemotaxis and chemoinvasionhave become available which employ a configuration in which two wellsare horizontally offset with respect to one another. This configurationof a device was introduced by Sally Zigmond in 1977 and, hereafterreferred to as the “Zigmond device,” consists of a 25 millimeters(mm)×75 mm glass slide with two grooves 4 mm wide and 1 mm deep,separated by a 1 mm bridge. One of the grooves is filled with anattractant and the other groove is filled with a control solution, thusforming a concentration gradient across the bridge. Cells are then addedto the other groove. Two holes are provided at each end of the slide toaccept pin clamps. The clamps hold a cover glass in place duringincubation and observation of the cells. Because of the size andconfiguration of the Zigmond chamber, it does not allow integration withexisting robotic liquid handling systems and automatic image acquisitionsystems. Further, as with transwell-based systems, the changes in cellmorphology during chemotaxis cannot be observed in real-time with theuse of the Zigmond chamber as the cells are fixed to a slide forobservation. In addition, the pin clamps must be assembled with an allenwrench and thus the device requires extra handling, positioning, andalignment before performing the assay. Such handling and positioning ofthe cover glass on the glass slide, as well as the rigidity of the coverglass, can potentially damage or interfere any surface treatment on thebridge.

[0012] A chemotaxis device attempting to solve the problem of lack ofreal-time observation is the “Dunn chamber.” The Dunn chamber consistsof a specially constructed microslide with a central circular sink and aconcentric annular moat. In an assay using a Dunn chamber, cells migrateon a coverslip, which is placed inverted on the Dunn chamber, towards achemotactic stimulus. The cells are monitored over-night using aphase-contrast microscope fitted with a video camera connected to acomputer with an image-grabber board. In addition to the problems ofrigidity of the coverslip and the lack of integration int o existingrobotic liquid handling systems, a major problem with the Dunn chamberassay is that only a very small number of cells are monitored (typicallyten). The average behavior of this very small sample may not be typicalof the population as a whole. A second major problem is that replicationis very restricted. Each control chamber and each treatment chamber mustbe viewed in separate microscopes, each one similarly equipped withcamera and computer.

[0013] Another chemotaxis device known in the art is disclosed in U.S.Pat. No. 6,238,874 to Jarnigan et. al. (the '874 patent). The '874patent discloses various embodiments of test devices that may be used tomonitor chemotaxis. However, disadvantageously, the devices in Jamaginet al. can not be easily sealed or assembled or peeled and disassembled.Thus, it is difficult to maintain surfaces that are prepared chemicallyor biologically during assembly. The test devices of the '874 patent aretherefore more suited for one-time use. Also, disassembly and collectionof cells is difficult to do without damage to the cells or withoutdisturbing the cell positions.

[0014] The prior art has failed to provide a test device, such as adevice for monitoring chemotaxis, haptotaxis, and/or chemoinvasion,which device is easily assembled and dissembled. In addition, the priorart has failed to provide a test device for monitoring chemotaxis and/orchemoinvasion, which is not limited to measuring the effects on cellmigration of chemoattractants, chemorepellants and chemostimulants.

SUMMARY OF THE INVENTION

[0015] The present invention provide a method of monitoring haptotaxis.The method includes providing a device for monitoring haptotaxis havinga housing defining a chamber. The chamber includes a first well regionincluding at least one first well, the first well region configured toreceive a test agent therein and further including biomoleculesimmobilized therein; a second well region including at least one secondwell, the second well region configured to receive a sample comprisingcells therein and further being horizontally offset with respect to thefirst well region in a test orientation of the device; and a channelregion with biomolecules immobilized therein and including at least onechannel connecting the first well region and the second well region withone another. The method further includes forming a surface concentrationgradient along a longitudinal axis of the chamber by decreasing theconcentration of biomolecules from the at least one first well to the atleast one second well. The method additionally includes placing a firstsample comprising cells in the at least one second well. The method alsoincludes monitoring haptotaxis of the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The various features of the invention will best be appreciated bysimultaneous reference to the description that follows and theaccompanying drawings, in which:

[0017]FIG. 1A is a top, perspective view, in partial cross section, of aportion of an embodiment of test device according to the presentinvention;

[0018]FIG. 1B is a top, perspective view of an embodiment of a testdevice of the present invention;

[0019]FIG. 1C is a side-elevational view of a longitudinal cross sectionof one of the chambers of the test device of FIG. 1B;

[0020]FIG. 2A is a schematic outline depicting a top plan view of analternative embodiment of a chamber defined in a test device of thepresent invention, where the channel region defines a single channel;

[0021]FIG. 2B is a schematic outline depicting a top plan view of theembodiment of the chambers defined in the embodiment of the test deviceaccording to FIG. 1B, where the channel region defines a single channel;

[0022]FIG. 2C is a figure similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the channel region defines a single channel;

[0023]FIG. 3A is a figure similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the channel region defines a plurality of channelshaving identical lengths;

[0024]FIG. 3B is a figure similar to FIG. 3A, showing a channel regiondefining a plurality of channels having lengths that increase from oneside of the chamber to another side of the chamber;

[0025]FIG. 3C is a figure similar to FIG. 3A, showing a channel regiondefining a plurality of channels having widths that increase from oneside of the chamber to another side of the chamber;

[0026]FIG. 4A is a figure similar to FIG. 1B showing an alternativeembodiment of a test device according to the present invention;

[0027]FIG. 4B is an enlarged, schematic, top plan view of a channel ofFIG. 4A showing cells on the sides of the channel;

[0028]FIGS. 5 and 6 are views similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the wells are trapezoidal in a top plan view thereof;

[0029]FIG. 7 is a view similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the chamber is in the form of a FIG. 8 in a top planview thereof;

[0030]FIG. 8 is a view similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where one well is rectangular and the other well circular ina top plan view of the device;

[0031]FIG. 9 is a view similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the first well region and the second well region eachdefine a plurality of wells, and where the channel region defines aplurality of channels joining respective wells of each well region;

[0032]FIG. 10 is a view similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the channel region defines a plurality of channelsjoining respective wells of each well region;

[0033]FIG. 11 is a view similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the first well region has a plurality of wells and arespective capillary for each well, the channel region has a singlechannel, and the second well region has a single well;

[0034]FIG. 12 is a side, cross-sectional view of an embodiment of aportion of the support member according to the present invention, theportion of the support member being shown along a longitudinal axis of achamber according to the present invention;

[0035]FIG. 13 is an isometric view of a collective system according toone embodiment of the present invention;

[0036]FIG. 14 is a view similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the first well region includes a plurality of wellsinterconnected by a network of capillaries, where the channel regionincludes a single channel, and where the second well includes a singlewell;

[0037]FIG. 15 is a block diagram of an automated analysis systemaccording to an embodiment of the present invention;

[0038]FIG. 16 is a flow diagram of a method according to an embodimentof the present invention;

[0039]FIG. 17 illustrates exemplary image data on which the method ofFIG. 16 may operate;

[0040]FIG. 18 illustrates a histogram that may be obtained from theimage data of FIG. 17;

[0041]FIG. 19 illustrates exemplary image data;

[0042]FIG. 20 is a histogram that may be obtained from exemplary imagedata of FIG. 19;

[0043]FIG. 21 illustrates exemplary dilated image data.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] As shown in FIG. 1A, according to one embodiment of the presentinvention, a test device 10, such as, for example, a cellularchemotaxis/haptotaxis and/or chemoinvasion device, includes a housing 10a comprising a support member 16 and a top member 11 mounted to thesupport member 16 by being placed in substantially fluid-tight,conformal contact with the support member 16. In the context of thepresent invention, “conformal contact” means substantially form-fitting,substantially fluid-tight contact. The support member 16 and the topmember 11 are configured such that they together define a discretechamber 12 as shown. Preferably test device 10 comprises a plurality ofdiscrete chambers, as shown by way of example in the embodiment of FIG.1B. The discrete chamber 12 includes a first well region 13 a includingat least one first well 13 and second well region 14 a including atleast one second well 14, the second well region further beinghorizontally offset with respect to the first well region in a testorientation of the device. The “test orientation” of the device is meantto refer to a spatial orientation of the device during testing. As shownin FIG. 1C, device 10 further includes a channel region 15 a includingat least one channel 15 connecting the first well region 13 a and thesecond well region 14 a with one another. In the embodiments of FIGS.1A-2C, each well region includes a single well, and the channel regionincludes a single channel. As seen in FIG. 1C, each well is defined by athrough-hole in top member 11, corresponding to well 13 and well 14respectively, and by an upper surface U of support member 16. Inparticular, the sides of each well 13 and 14 are defined by the walls ofthe through holes in the top member 11, and the bottoms of well 13 and14 are defined by the upper surface U of support member 16. It is notedthat in the context of the present invention, “top,” “bottom,” “upper”and “side” are defined relative to the test orientation of the device.As seen collectively in FIGS. 1A and 1C, a length L of channel region 15a is defined in a direction of the longitudinal axis of channel region15 a; a depth D of channel region 15 a is defined in a direction normalto upper surface U of support member 16; a width W is defined in adirection normal to the length L and depth D of channel region 15 a.According to one embodiment of the present invention, the chamber'sfirst well 13 is adapted to receive a test agent, a soluble testsubstance and/or a test agent comprising immobilized biomolecules, whichpotentially affects chemotaxis or haptotaxis. Biomolecules include, butnot limited to, DNA, RNA, proteins, peptides, carbohydrates, cells,chemicals, biochemicals, and small molecules. The chamber's second well14 is adapted to receive a biological sample of cells. Immobilizedbiomolecules are biomolecules that are attracted to support member 16with an attractive force stronger than the attractive forces that arepresent in the environment surrounding the support member, such assolvating and turbulent forces present in a fluid medium. Non-limitingexamples of the test agent include chemorepellants, chemotacticinhibitors, and chemoattractants, such as growth factors, cytokines,chemokines, nutrients, small molecules, and peptides. Alternatively, thechamber's first well 13 is adapted to receive a biological sample ofcells and the chamber's second well 14 is adapted to receive a testagent.

[0045] In one embodiment of the present invention, when a soluble testsubstance is used as the test agent, channel region 15 a preferablycontains a gel matrix. The gel matrix allows the formation of a solutionconcentration gradient from first well region 13 a towards second wellregion 14 a as the solute diffuses from an area of higher concentration(well region 13 a) through a semi-permeable matrix (the gel matrix) toan area of lower concentration (well region 14 a). If the soluble testsubstance comprises a chemoattractant, in order for the cells to migratethrough the matrix in the direction of the solution concentrationgradient towards well region 13 a, the cells must degrade this matrix byreleasing enzymes such as matrix metalloproteases. This cell chemotaxisand invasion may be subsequently observed, measured, and recorded.

[0046] In one embodiment of the present invention, utilizing immobilizedbiomolecules as the test agent, the biomolecules are preferablyimmobilized or bound on the portion of support member 16 underlyingchannel region 15 a and underlying through hole for well region 13 a.The concentration of biomolecules decreases along the longitudinal axisof the device from well region 13 a towards well region 14 a forming asurface concentration gradient of immobilized biomolecules and thebiological sample of cells potentially responds to this surfacegradient. This cell haptotaxis may be subsequently observed, measured,and recorded.

[0047] With respect to particular specifications of device 10, topmember 11 is made of a material that is adapted to effect conformalcontact, preferably reversible conformal contact, with support member16. According to embodiments of the present invention, the flexibilityof such a material, among other things, allows the top member toform-fittingly adhere to the upper surface U of support member 16 insuch a way as to form a substantially fluid-tight seal therewith. Theconformal contact should preferably be strong enough to prevent slippageof the top member on the support member surface. Where the conformalcontact is reversible, the top member may be made of a material havingthe structural integrity to allow the top member to be removed by asimple peeling process. This would allow top member 11 to be removed andcells at certain positions collected. Preferably, the peeling processdoes not disturb any surface treatment or cell positions of supportmember 16. Physical striations, pockets, SAMs, gels, peptides,antibodies, or carbohydrates can be placed on support member 16 and thetop member 11 subsequently can be placed over support member 16 withoutany damage to these structures. Additionally, the substantiallyfluid-tight seal effected between top member 11 and support member 16 byvirtue of the conformal contact of top member 11 with support member 16prevents fluid from leaking from one chamber to an adjacent chamber, andalso prevents contaminants from entering the wells. The seal preferablyoccurs essentially instantaneously without the necessity to maintainexternal pressure. The conformal contact obviates the need to use asealing agent to seal top member 11 to support member 16. Althoughembodiments of the present invention encompass use of a sealing agent,the fact that such a use is obviated according to a preferred embodimentprovides a cost-saving, time-saving alternative, and further eliminatesa risk of contamination of each chamber 12 by a sealing agent.Preferably, the top member 11 is made of a material that does notdegrade and is not easily damaged by virtue of being used in multipletests, and that affords considerable variability in the top member'sconfiguration during manufacture of the same. More preferably, thematerial may be selected for allowing the top member to be made usingphotolithography. In a preferred embodiment, the material comprises anelastomer such as silicone, natural or synthetic rubber, orpolyurethane. In a more preferred embodiment, the material ispolydimethylsiloxane (“PDMS”).

[0048] According to a preferred embodiment of a method of the presentinvention, standard photolithographic procedures can be used to producea silicon master that is the negative image of any desired configurationof top member 11. For example, the dimensions of chambers 12, such asthe size of well regions 13 a and 14 a, or the length of channel region15 a, can be altered to fit any advantageous specification. Once asuitable design for the master is chosen and the master is fabricatedaccording to such a design, the material is either spin cast, injected,or poured over the master and cured. Once the mold is created, thisprocess may be repeated as often as necessary. This process not onlyprovides great flexibility in the top member's design, it also allowsthe top members to be massively replicated. The present invention alsocontemplates different methods of fabricating device 10 including, forexample, e-beam lithography, laser-assisted etching, and transferprinting.

[0049] Device 10 preferably fits in the footprint of an industrystandard microtiter plate. As such, device 10 preferably has the sameouter dimensions and overall size of an industry standard microtiterplate. Additionally, when chamber 12 comprises a plurality of chambers,either the chambers 12 themselves, or the wells of each chamber 12, mayhave the same pitch of an industry standard microtiter plate. The term“pitch” used herein refers to the distance between respective verticalcenterlines between adjacent chambers or adjacent wells in the testorientation of the device. The embodiment of device 10, shown in FIG.1B, comprises 48 chambers designed in the format of a standard 96-wellplate, with each well fitting in the space of each macrowell of theplate. The size and number of the plurality of chambers 12 cancorrespond to the footprint of standard 24-, 96-, 384-, 768- and1536-well microtiter plates. For example, for a 96 well microtiterplate, device 10 may comprise 48 chambers 12 and therefore 48experiments can be conducted, and for a 384 well microtiter plate, thedevice may comprise 192 chambers 12, and therefore 192 experiments canbe conducted. The present invention also contemplates any other numberof chambers and/or wells disposed in any suitable configuration. Forexample, if pitch or footprint standards change or new applicationsdemand new dimensions, then device 10 may easily be changed to meetthese new dimensions. By conforming to the exact dimension andspecification of standard microtiter plates, embodiments of device 10would advantageously fit into existing infrastructure of fluid handling,storage, registration, and detection. Embodiments of device 10,therefore, may be conducive to high throughput screening as they mayallow robotic fluid handling and automated detection and data analysis.Top member 11 may additionally take on several different variations andembodiments. Depending on the test parameters, such as, for example,where chemotaxis, haptotaxis and/or chemoinvasion are to be monitored,the cell type, cell number, or distance over which chemotaxis orhaptotaxis is required, chamber 12 of top member 11 may have variousembodiments of which a few exemplary embodiments are discussed herein.With respect to a discrete chamber 12, the shape, dimensions, location,surface treatment, and numbers of channels in channel region 15 a andthe shape and number of wells 13 and 14 may vary.

[0050] Regarding the shape of channel region 15 a, each channel 15 inthe channel region 15 a is not limited to a particular cross-sectionalshape, as taken in a plane perpendicular to its longitudinal axis. Forexample, the cross section of any given channel 15 can be hexagonal,circular, semicircular, ellipsoidal, rectangular, square, or any otherpolygonal or curved shape.

[0051] Regarding the dimensions of a channel 15, the length L of a givenchannel 15 can vary based on various test parameters. For instance, thelength L of a given channel 15 may vary in relation to the distance overwhich chemotaxis or haptotaxis is required to occur. For example, thelength L of a given channel 15 can range from about 3 μm to about 18 mmin order to allow cells sufficient distance to travel and thereforesufficient opportunity to observe cell chemotaxis/haptotaxis andchemoinvasion. The width W and depth D of a given channel 15 may alsovary as a function of various test parameters. For examples, the width Wand depth D of a given channel 15 may vary, in a chemotaxis, haptotaxisand/or chemoinvasion device, depending on the size of the cell beingstudied and whether a gel matrix is added to the given channel 15.Generally, where the test device is a chemotaxis, haptotaxis and/orchemoinvasion device, a given channel 15's width W and depth D may beapproximately in the range of the diameter of the cell being assayed. Todiscount random cellular movement, at least one of the depth D or widthW of a given channel 15 should preferably be smaller than the diameterof the cell when no gel matrix is placed in the given channel 15 so thatwhen the cells are activated, they can “squeeze” themselves through thegiven channel toward the test agent. If a given channel 15 contains agel matrix, then, the depth D and width W of the given channel 15 may begreater than the diameter of the cell being assayed. Referring by way ofexample to the embodiments of FIGS. 1A-2C, if suspension cells such asleukocytes, which are about 3-12 μm in diameter, are in well 14 andchannel 15 contains no gel, then the width W of channel 15 should rangefrom about 3 microns to about 20 μm, and the depth D of channel 15should range from about 3 microns to about 20 μm but at least either thedepth D or width W of channel 15 should be smaller than the diameter ofthe cell. If leukocytes are in well 14 and channel 15 contains a gelmatrix, then the width W of channel 15 should range from about 20 toabout 100 μm and the depth D should range from about 20 μm to about 100μm, and both the width W and depth D of channel 15 can be greater thanthe diameter of the cell assayed. Similarly, if adherent cells, such asendothelial cells which are 3-10 microns in diameter before adherence,are in well 14 and channel 15 contains no gel, then the width W anddepth D of channel 15 can range from about 3 to about 20 μm, but atleast either the width W or depth D of channel 15 should be smaller thanthe diameter of the cell assayed. If adherent cells are in well 14 andchannel 15 contains a gel matrix then the width W and depth D of channel15 should range from about 20 μm to about 200 μm and both the width Wand depth D of channel 15 can be greater than the diameter of the cellassayed.

[0052] As seen in FIGS. 2A-2C channel 15 may connect the first well 13to the second well 14 at respective sides of the wells, as shown inFIGS. 2A and 2C or at a central region of the wells, as shown in FIG.2B.

[0053] The number of channels in channel region 15 a between wellregions 13 a and 14 a can also vary. Channel region 15 a may include aplurality of channels, as shown by way of example in FIGS. 3A-3C. Asseen in FIG. 3A, in a preferred configuration, the length L of eachchannel 15 i-n between well 13 and well 14 is identical. In anotherembodiment as seen in FIG. 3B, the length L of each channel 15 i-15 n ofchannel region 15 a increases in the direction of well 14, starting fromchannel 15 i in the side portion 12 a of chamber 12 to channel 15 n inthe side portion 12 b of chamber 12. In one embodiment, as seen in FIG.3B, the length L of each successive channel in the plurality of channels15 of chamber 12 increases in a direction of a width W of the channelswith respect to a preceding one of the plurality of channels such thatrespective channel inlets at one of the first well region and the secondwell region, such as well region 13 a as shown, are aligned along thedirection of the width W of the channels. Although, in thisconfiguration, the cells traveling in any particular channel will exitthe channels and enter well 14 at points longitudinally offset withrespect to one another, the section of channel region 15 a closest towell region 13 a is positioned so that cells ultimately entering thedifferent channels will be aligned in a direction of the width W of thechannels so that there is no longitudinal offset between them.Therefore, in comparing two adjacent channels, a first group of cellsentering channel 15 i has an entry position that is not longitudinallyoffset with respect to a second different group of cells enteringchannel 15 j, but the first group of cells exiting channel 15 i has anexit point longitudinally offset from the second group of cells exitingchannel 15 j. In a different embodiment of the present inventionillustrated in FIG. 3C, the width W of each channel 15 i-15 n increasesstarting from channel 15 i in the side portion 12 a of chamber 12 tochannel 15 n in the side portion 12 b of chamber 12. Preferably, thewidth W or depth D of each successive channel of the plurality ofchannels increases in a direction of a width W of the channels withrespect to a preceding one of the plurality of channels. Alternatively,a depth D of each successive channel could increase (not shown) along adirection of the width W of the channels. It is understood to thoseskilled in the art, that various embodiments altering the dimensions ofthe channels in the channel region 15 a are within the scope of thepresent invention. For example, the length of the channels 15 i-15 nneed not increase in a continuous manner from channel 15 i to 15 n asillustrated in FIG. 3B. Instead, channel 15 i-15 n may have varyinglengths following no particular order or pattern.

[0054] With respect to surface treatment of a given channel 15, tosimulate in vivo conditions where cells are surrounded by other cells,the lateral walls of a given channel 15 may be coated with cells, suchas endothelial cells 40 as seen in FIG. 4B. Non-limiting examples ofendothelial cells include human umbilical vein endothelial cells or highendothelial venule cells. In another embodiment, a given channel 15 isfilled with a gel matrix such as gelatin, agarose, collagen, fibrin, anynatural or synthetic extracellular proteinous matrix or basal membranemimic including, but not limited to MATRIGEL™ (Becton DickensonLabware), or ECM GEL, (Sigma, St. Louis, Mo.), or other hydrogelscontaining certain factors such as cytokines, growth factors,antibodies, ligands for cell surface receptors, or chemokines.Preferably, the gel has a substantially high water content and is porousenough to allow cell chemotaxis and invasion. As mentioned above, whenthe test agent comprising a soluble test substance is placed in well 13,the gel facilitates formation of a solution concentration gradient alongthe longitudinal axis of chamber 12. Additionally, adding a gel matrixto a given channel 15 simulates the natural environment in the body, asenzyme degradation through extracellular matrix is a crucial step in theinvasive process.

[0055] According to the present invention, the individual wells of eachwell region 13 a or 14 a may have any shape and size. For example, thetop plan contour of a given well may be circular, as shown in FIGS.1A-2C, or trapezoidal as shown in FIGS. 5 and 6. Alternatively, the topplan contour of a given chamber may be generally in the shape of a“figure 8” as shown in FIG. 7. Preferably when using a soluble testsubstance as the test agent, the shape of well 13 is such that solubletest substance is readily able to access the channel 15 and thereby formthe necessary solution concentration gradient along the length L ofchannel 15. Preferably, the shape of well 14 is such that cells are notdeferred, detained, or hindered from migrating out of the first well 14,for example, by accumulating in a comer, side or other dead space ofwell 14. Although possible accumulation of cells in a dead space of well14 is not restricted to any particular cell number, there exists agreater likelihood of cells accumulating in a corner of well 14 if alarge number of cells are used. Therefore to maximize accessibility tothe concentration gradient and to minimize the “wasting” of cells when alarge cell sample is utilized, it is important that the shape of well 14be such that a sufficiently high percentage of cells, particularly thecells in the area of well 14 furthest from channel 15, are capable ofmigrating out of well 14. In a different embodiment that also addressesthe problem of the wasting of cells, well 14 may be shaped such that allcells have a higher probability of accessing the concentration gradient.For example as seen in FIG. 8, the length L_(w) of well 14 in a top planview thereof is minimized to decrease the surface area of the well. Assuch, the cells are closer to the concentration gradient formed inchannel 15. In a preferred embodiment, the L_(w) of well 14 in a topplan view thereof is about 1 mm to about 2 mm.

[0056] In addition to variations of components of a discrete chamber 12,the present invention also contemplates variations in the overallchamber 12 as well as variations from chamber to chamber. With respectto the overall chamber 12, in one embodiment, the chambers 12 are sizedso that a complete chamber 12 fits into the area normally required for asingle well of a 96-well plate. In this configuration, 96 differentassays could be performed in a 96-well plate. In another embodiment, the1:1 ratio of a first well to second well, as present in theaforementioned embodiments, is altered by modifying chamber 12. Forexample as seen in FIG. 9, device 10 includes a chamber 12 having afirst well region 13 a having a plurality of first wells 102, 103, 104and 105 connected to one another, a second well region 14 a having aplurality of wells 106, 107, 108 and 109, and a channel region 15 ahaving a plurality of channels 15 connecting respective ones of thefirst wells to respective ones of the second wells. Each well of thefirst well region 13 a may receive the same test agent, and each well ofthe second well region 14 a may receive a different cell type.Alternatively, each well of the first well region 13 a may receive adifferent test agent, and each well of the second well region 14 a mayreceive the same cell type. This configuration allows several differentcell types or different test agents to be tested simultaneously. In analternative embodiment as seen in FIG. 10, each channel 15 of channelregion 15 a comprises subchannels as shown. This arrangement not onlyallows several different cell types or test agents to be testedsimultaneously but also generates several tests of each test agent orcell type.

[0057]FIG. 11 illustrates an alternative chamber configuration of a testdevice according to an alternative embodiment of the present invention.In this embodiment, chamber 12 comprises a first well region 13 aconnected by a channel region 15 a including a single channel 15 to asecond well region 14 a including a single well 14. The first wellregion contains a plurality of first wells, 17 a, 18 a, and 19 a and aplurality of capillaries, a first perimeter capillary 17, a centercapillary 18, and a second perimeter capillary 19 connected torespective ones of the plurality of first wells. All three of thecapillaries converge at a junction into channel 15, which is connectedwith the second well region 14 a. Well region 13 a is not limited tocontaining only three capillaries and can contain any number ofadditional capillaries (not shown). First wells 17 a-19 a may, forexample, be adapted to receive solutions of biomolecules, which areallowed to flow into channel 15 and adsorb nonspecifically to theregions of the surface over which the solution containing thebiomolecules flows. First wells 17 a-19 a are also adapted tosubsequently receive cells. With respect to variations from chamber tochamber, in one embodiment, the length L of each channel 15 increasesalong one or more dimensions of top member 11 from one chamber to theadjacent chamber. In an alternative embodiment, all chambers 12 havechannel 15 of the same length L. The width W of each channel 15 can alsovary and can increase along one or more dimensions of top member 11 fromone chamber to the adjacent chamber. In an alternative embodiment, allchambers 12 have channel 15 of the same width W. FIG. 4A is a top planview of an embodiment of the present invention where, within top member11, different chambers have various channel sizes and shapes, such sizesand shapes being in no particular order, pattern, or arrangement. Byemploying this varied configuration, the best channel region design fora given test may be obtained. In other words, where the optimal channelregion design is determined, a new assay plate configured solely tothose specifications may be employed.

[0058] Support member 16 of device 10 provides a support upon which topmember 11 rests and can be made of any material suitable for thisfunction. Suitable materials are known in the art such as glass,polystyrene, polycarbonate, PMMA, polyacrylates, and other plastics.Where device 10 is a chemotaxis, haptotaxis and/or chemoinvasion device,it is preferable that support member 16 comprise a material that iscompatible with cells that may be placed on the surface of supportmember 16. Suitable materials may include standard materials used incell biology, such as glass, ceramics, metals, polystyrene,polycarbonate, polypropylene, as well as other plastics includingpolymeric thin films. A preferred material is glass with a thickness ofabout 0.1 to about 2 mm, as this may allow the viewing of the cells withoptical microscopy techniques.

[0059] Similar to top member 11, support member 16 can have severaldifferent embodiments. In particular, the configuration and surfacetreatment of support member 16 may vary.

[0060] As seen in a side view of support member 16 in FIG. 12, the uppersurface U of support member 16, which underlies top member 11, may besloped at predetermined regions thereof with respect to a horizontalplane at less than a 90° angle. In the shown embodiment, thepredetermined regions correspond to bottom surfaces of respective wells,surface 16 a corresponding to a bottom surface of a well 13, and surface16 b corresponding to a bottom surface of well 14. Surface 16 c, inturn, corresponds to a bottom surface of channel 15. In this embodiment,the given configuration facilitates suspended cells flowing in thedirection of the downward slope of top surface 16 b of support member 16to become more readily exposed to the concentration gradient. If asoluble test substance is used as the test agent in well 13 of device10, then top surface 16 a of support member 16 may also be downwardlysloped with respect to a horizontal plane at less than a 90° angle tofacilitate exposure of the test substance to channel 15 in order tofacilitate formation of the solution concentration gradient.

[0061] Support member 16 may also have a treatment on or embedded intoits surface. This treatment may serve numerous functions, including, forexample, facilitating the placement, adhesion or movement of cells beingstudied, and simulating in vivo conditions. Numerous surfaceconfigurations and chemicals may be used alone or in conjunction forthis treatment. For example, in one embodiment support member 16includes a patterned self-assembled monolayer (SAM) on a gold surface orother suitable material. SAMs are monolayers typically formed ofmolecules each having a functional group that selectively attaches to aparticular surface, the remainder of each molecule interacting withneighboring molecules in the monolayer to form a relatively orderedarray. By using SAMs, various controls of biological interactions may beemployed. For example, SAMs may be arrayed or modified with various“head groups” to produce “islands” of biospecific surfaces surrounded byareas of bio-inert head groups. Further, SAMs may be modified to have“switchable surfaces” that may be designed to capture a cell and then besubsequently modified to release the captured cell. Moreover, it mayalso be desirable to utilize a bioinert support member material toresist non-specific adsorption of cells, proteins, or any otherbiological material. Consequently, the use of SAMs on support member 16may be advantageous.

[0062] The present invention also contemplates, as seen in FIG. 13, theuse of any system known in the art to detect and analyze cellchemotaxis, haptotaxis, and chemoinvasion. In particular, the presentinvention contemplates the use of any system known in the art tovisualize changes in cell morphology as cells move across channel 15, tomeasure the distance cells travel in channel 15, and to quantify thenumber of cells that travel to particular points in channel 15. As suchthe present invention contemplates both “real-time” and “endpoint”analysis of chemotaxis, haptotaxis, and chemoinvasion. In oneembodiment, the device 122 includes an observation system 120 and acontroller 121. The controller 121 is in communication with theobservation system 120 via line 122. The controller 121 and observationsystem 120 may be positioned and programmed to observe, record, andanalyze chemotaxis and chemoinvasion of the cells in the device. Theobservation system 120 may be any of numerous systems, including amicroscope, a high-speed video camera, and an array of individualsensors. Nonlimiting examples of microscopes include phase-contrast,fluorescence, luminescence, differential-interference-contrast, darkfield, confocal laser-scanning, digital deconvolution, and videomicroscopes. Each of these embodiments may view or sense the movementand behavior of the cells before, during, and after the test agent isintroduced. At the same time, the observation system 120 may generatesignals for the controller 121 to interpret and analyze. This analysiscan include determining the physical movement of the cells over time aswell as their change in shape, activity level or any other observablecharacteristic. In each instance, the conduct of the cells being studiedmay be observed in real time, at a later time, or both. The observationsystem 120 and controller 121 may provide for real-time observation viaa monitor. They may also provide for subsequent playback via a recordingsystem of some kind either integrated with these components or coupledto them. For example, in one embodiment, cell behavior during thedesired period of observation is recorded on VHS format videotapethrough a standard video camera positioned in the vertical ocular tubesof a triocular compound microscope or in the body of an invertedmicroscope and attached to a high quality video recorder. The videorecorder is then played into a digitization means, e.g., PCI framegrabber, for the conversion of analog data to digital form. Theelectronic readable (digitized) data is then accessed and processed byan appropriate dynamic image analysis system, such as that disclosed inU.S. Pat. No. 5,655,028 expressly incorporated in its entirety herein byreference. Such a system is commercially available under the trademarkDIAS® from Solltech Inc. (Oakland, Iowa). Software capable of assistingin discriminating cells from debris and other detection artifacts thatmight be present in the sample should be particularly advantageous. Ineither case, these components may also analyze the cells as theyprogress through their reaction to the test agent.

[0063] In one embodiment, the present invention contemplates the use ofan automated analysis system, as illustrated in FIG. 15, to analyze datameasuring the distance cells travel in channel 15, and to quantify thenumber of cells that travel to particular points in channel 15. FIG. 15is a block diagram of an automated analysis system 100 including, forexample, an image preprocessing stage 110, an object identificationstage 120 and a migration analysis stage 130. The image preprocessingstage 110 may receive digital image data of chamber 12 from a digitalcamera or other imaging apparatus as described above. The data typicallyincludes a plurality of image samples at various spatial locations(called, “pixels” for short) and may be provided as color or grayscaledata. The image preprocessing stage 110 may alter the captured imagedata to permit algorithms of the other stages to operate on it. Theobject identification stage 120 may identify objects from within theimage data. Various objects may be identified based on the test to beperformed. For example, the object identifier may identify channels 15,cells or cell groups from within the image data. The migration analysisstage 130 may perform the migration analysis designated for testing.FIG. 15 illustrates a number of blocks that may be included within theimage preprocessing stage 110. Essentially, the image preprocessingstage 110 counteracts image artifacts that may be present in thecaptured image data as a result of imperfections in the imager or thedevice. In one embodiment, the image preprocessing stage 110 may includean image equalization block 140. The equalization 140 may findapplication in embodiments where sample values of captured image data donot occupy the full quantization range available for the data. Forexample, an 8-bit grayscale system permits 256 different quantizationlevels for input data (0-255). Due to imperfections in the imagingprocess, it is possible that pixel values may be limited to a narrowrange, say the first 20 quantization levels (0-20). The equalization 140may re-scale sample values to ensure that they occupy the full rangeavailable in the 8-bit system.

[0064] In another embodiment, the equalization block 140 may re-scalesample values based on a color or wavelength. Conventional cellularanalysis techniques often cause cells to appear in predetermined colorsor with predetermined wavelengths, which permits them to bedistinguished from other materials captured by the imager. For example,in fluorescent applications, cells emit light at predeterminedwavelengths. In nuclear staining applications, cell nuclei are dyed witha material that causes them to appear in the image data withpredetermined colors. The equalization block 140 may re-scale samplevalues having components that coincide with these expected colors orwavelengths. In so doing, the equalization block 140 effectively filtersout other colors or wavelengths, a consequence that may be advantageousin later image processing.

[0065] Image rotation is another image artifact that may occur fromimperfect imaging apparatus. Although the channels 15 are likely to begenerally aligned with columns and rows of pixels in the image data,further analysis may be facilitated if the alignment is improved.Accordingly, in an embodiment, the image preprocessing stage 110 mayinclude an image alignment block 150 that rotates the captured imagedata to counteract this artifact. Once the rotation artifact has beenremoved from the captured image data, then image from individualchannels 15 are likely to coincide with a regular row or column array ofpixel data. FIG. 16 illustrates a method of operation for the imagealignment block 150 according to an embodiment of the present inventionand described in connection with exemplary image data illustrated inFIG. 17. In the example of FIG. 17, channels 15 are aligned generallywith rows of image data but for the rotation artifact. To counteract therotation artifact, the image preprocessor may identify a band of imagedata coinciding with a boundary between second well 14 and the channels15 themselves (block 1010). In the case of FIG. 17, the band mayconstitute column 310. Generally, the area of second well 14 will bebright relative to the area of channels 15 due the greater number ofcells present therein. Thus, a histogram of image data values along apresumed direction of the channels 15 may appear as shown in FIG. 18.The band 310 may be identified from an abrupt change in image datavalues along this direction.

[0066] Having identified a column of image data to be considered, thecolumn 310 may be split into two boundary boxes 320, 330 (block 1020).By summing the intensity of the image data in each of the two boundaryboxes and comparing summed values to each other, an orientation of therotation artifact may be determined (blocks 1030, 1040). In the exampleof FIG. 17, the rotation artifact causes more of second well 14 to fallwithin the area of boundary box 320 than of boundary box 330 (aclockwise artifact). The image data may be rotated counterclockwiseuntil the summed values of each boundary box 320, 330 become balanced.

[0067] Thus, if the image intensity of the first bounding box is greaterthan that of the second bounding box 330, the image data may be rotatedin a first direction (block 1050). If the image intensity of the secondbounding box 330 is greater than that if the first bounding box 320, theimage data may be rotated in a second direction (block 1060). And whenthe image intensities are balanced, the method 1000 may conclude; therotation artifact has been corrected.

[0068] Returning to FIG. 15, the image preprocessing stage 110 also mayprocess the captured image data by cropping the image to the areaoccupied by channels 15 themselves (block 160). As described, each testbed may include a pair of wells interconnected by a plurality ofchannels. For much of the migration analysis, it is sufficient tomeasure cellular movement or activity within channels 15 only. Activityin second well 14 or the first well 13 need not be considered. In suchan embodiment, the image preprocessing stage 110 may crop the image datato remove pixels that lie outside channels 15.

[0069] The image preprocessing stage 110 also may include a thresholdingblock 170, performing threshold detection upon the image data. Thethresholding block 170 may truncate to zero any sample having are-scaled value that fails to exceed a predetermined threshold. Suchthresholding is useful to remove noise from the captured image data. Inan embodiment, the thresholding block 170 may be integrated with theequalization block 140 discussed above. It need not be present as aseparate element. In some embodiments, particularly those where theequalization block 140 scales pixel values according to wavelengthcomponents, the thresholding block 170 may be omitted altogether. Anoutput of the image preprocessing stage 110 may be input to the objectidentification stage 120. The object identification stage 120 identifiesobjects from within the image data, including the microchannelsthemselves and, optionally, individual cells. According to anembodiment, in a fluorescent system, channels 15 may be identified bydeveloping a histogram of the fluorescent light along a major axis inthe system (block 180). FIG. 19 illustrates image data that may havebeen determined from the example of FIG. 17. The major axis may coincidewith the boundary between the second well adapted to receive a samplecomprising cells and the channel region. Light intensity from withinchannel region 15 a area may be summed along this axis, yielding a dataset represented in FIG. 19. In a second stage, the data set is “dilated”(block 190). Dilation may be achieved by applying a high pass filter tothe data set or any other analogous technique. FIG. 20 illustrates thedata set of FIG. 19 having been subject to dilation.

[0070] From the data set of FIG. 20, the channels may be identified.Candidate channel 15 positions may be identified to coincide withrelative maximums of the data set. Alternatively, candidate positions ofboundaries between channels 15 may be determined from relative minimumsfrom within the data set of FIG. 20. A final set of channel 15 positionsmay be determined from a set of parameters known about channel region 15a itself. For example, if channels 15 are known to have been providedwith a regular spacing among channels 15, any candidate channel 15position that would violate the spacing can be eliminated fromconsideration.

[0071] Returning to FIG. 15, in addition to identifying channels 15,individual cells may be identified within the image data (block 200). Inan application where cells are marked with nuclear staining,identification of individual cells merely requires an image processor toidentify and count the number of marked nuclei. The nuclei appear is anumber of dots of a predetermined color. In an application usingfluorescing cells, identification of individual cells becomes morecomplicated. Individual cells can be identified relatively easily; theyappear as objects of relatively uniform area in the image data.Identifying a number of cells clustered together becomes more difficult.In this case, the number of cells may be determined from the area orradius of the cluster in the image data. The cluster is likely to appearin the image having some area or cluster radius. By comparing thecluster's area or radius to the area or radius of an individual cell,the number of cells may be interpolated. Of course, identification ofindividual cells may be omitted depending upon the requirements of themigration analysis.

[0072] The final stage in the image processing system is the migrationanalysis 130 itself. In one embodiment, coordinate data of each cell inthe channels 15 may be gathered and recorded. However, some testing neednot be so complicated. In a first embodiment, it may be sufficientmerely to identify the number of cells present in channel 15. In thiscase, identification of individual cells may be avoided by merelysumming quantities of fluorescent light detected in each channel 15.From this measurement, the number of cells may be derived withoutinvesting the processing expense of identifying individual cells. Theforegoing description presents image analysis that is relevant to asingle channel 15 to be tested. Of course, depending upon therequirements of the migration analysis 130, it may be desired togenerate image samples of a number of different channels 15. Further, itmay be desirable to generate image samples of a single channel 15 atdifferent times. The image processing described above may be repeatedfor different channels 15 and different times to accommodate for suchtest scenarios.

[0073] According to an embodiment, the image processing may account formanufacturing defects of individual channels 15. During imageprocessing, manufacturing defects may prevent cell migrations into achannel 15. In an embodiment, when the system 100 counts a number ofcells in the channel 15 (or derives the number from identified celllocations), it may compare the number to an expectation threshold. Ifthe number is below the expectation threshold, the system 100 mayexclude the channel 15 from migration analysis. In practice, thisexpectation threshold may be established as a minimum number of cellsthat are likely to enter a properly configured cell given the testconditions being analyzed under the migration analysis. If the actualnumber of cells falls below this threshold, it may lead to a conclusionthat channel 15 blocking conditions may be present.

[0074] The foregoing operations and processes of the analysis system 100may be performed by general purpose processing apparatus, such ascomputers, workstations or servers, executing software. Alternatively,some of the operations or processes may be provided in a digital signalprocessor or application specific integrated circuit (colloquially, an“ASIC”). Additionally, these operations and processes, particularlythose associated with image preprocessing, may be distributed inprocessors of a digital microscope system. Such variations are fullywithin the scope of the present invention.

[0075] The present invention also contemplates the use of theaforementioned embodiments of device 10 to assay various elements ofchemotaxis, haptotaxis and chemoinvasion. In general, the presentinvention provides for a first assay comprising high throughputscreening of test agents to determine whether they influence chemotaxis,haptotaxis, and chemoinvasion. Test agents generally comprise eithersoluble test substances or immobilized test biomolecules and aregenerally placed in first well region 13 a of chamber 12 of device 10.After determining which test agents influence chemotaxis, by acting aschemoattractants and promoting or initiating chemotaxis, by acting aschemorepellants and repelling chemotaxis, or by acting as inhibitors andhalting or inhibiting chemotaxis, then a second assay can be performedscreening test compounds. The test compounds generally comprisetherapeutics or chemotaxis/haptotaxis inhibitors and are generallyintroduced in second well region 14 a, which contains a biologicalsample of cells. The test compounds are screened to determine if and howthey influence the cells' chemotaxis or haptotaxis in response to thetest agents.

[0076] In particular, a chemotaxis/haptotaxis and/or chemoinvasion assayaccording to an embodiment of the present invention involves a device 10including a housing comprising a top member 11 mounted to a supportmember 16. The top member and the support member are configured suchthat they together define a discrete assay chamber 12. The discreteassay chamber 12 includes a first well region 13 a connected by achannel 15 to a second well region 14 a. The first well region 13 aincludes at least one first well 13, each of the at least one first well13 being adapted to receive a test agent therein. The second well region14 a includes at least one second well 14 horizontally offset withrespect to the first well region 13 a in a test orientation of thedevice, each of the at least one second well 14 being adapted to receivea cell sample therein. Channel 15 includes at least one channelconnecting the first well region 13 a and the second well region 14 a toone another. The test agent received in first well 13 is a soluble testsubstance and/or immobilized test biomolecules. When the test agentcomprises immobilized test biomolecules, the biomolecules areimmobilized on an upper surface U of support member 16 constituting thebottom surface of well region 13 a as well as on upper surface U ofsupport member 16 constituting the bottom surface of channel region 15a.

[0077] Nonlimiting examples of biological samples of cells includelymphocytes, monocytes, leukocytes, macrophages, mast cells, T-cells,B-cells, neutrophils, basophils, eosinophils, fibroblasts, endothelialcells, epithelial cells, neurons, tumor cells, motile gametes, motileforms of bacteria, and fungi, cells involved in metastasis, and anyother types of cells involved in response to inflammation, injury, orinfection. Well region 14 a may receive only one cell type or anycombination of the above-referenced exemplary cell types. For example,as described above, it is often desirable to provide a mixed cellpopulation to more effectively create an environment similar to in vivoconditions. Well region 14 a may also receive cells at a particular cellcycle phase. For example, well region 14 a may receive lymphocytes in G₁phase or G₀ phase.

[0078] Nonlimiting examples of soluble test substances includechemoattractants, chemorepellants, or chemotactic inhibitors. Asexplained above, chemoattractants are chemotactic substances thatattract cells and once placed in well region 14 a, cause cells tomigrate towards well region 14 a. Chemorepellents are chemotacticsubstances that repel cells and once placed in well region 14 a, causecells to migrate away from well region 14 a. Chemotactic inhibitors arechemotactic substances that inhibit or stop chemotaxis and once placedin well region 14 a, cause cells to have inhibited migration or nomigration from well region 14 a. Non-limiting examples ofchemoattractants include hormones such as T₃ and T₄, epinephrine andvasopressin; immunological agents such as interleukein-2, epidermalgrowth factor and monoclonal antibodies; growth factors; peptides; smallmolecules; and cells. Cells may act as chemoattractants by releasingchemotactic factors. For example, in one embodiment, a sample includingcancer cells may be added to well 13. A sample including a differentcell type may be added to well 14. As the cancer cells grow they mayrelease factors that act as chemoattractants attracting the cells inwell 14 to migrate towards well 13. In another embodiment, endothelialcells are added to well 13 and activated by adding a chemoattractantsuch as TNF-α or IL-1 to well 13. Leukocytes are added to well 14 andmay be attracted to the endothelial cells in well 14.

[0079] Non-limiting examples of chemorepellants include irritants suchas benzalkonium chloride, propylene glycol, methanol, acetone, sodiumdodecyl sulfate, hydrogen peroxide, 1-butanol, ethanol, anddimethylsulfoxide; and toxins such as cyanide, carbonylcyanidechlorophenylhydrazone, endotoxins and bacterial lipopolysaccharides;viruses; pathogens; and pyrogens.

[0080] Nonlimiting examples of immobilized biomolecules includechemoattractants, chemorepellants, and chemotactic inhibitors asdescribed above. Further non-limiting examples of immobilizedchemoattactants include chemokines, cytokines, and small molecules.Further non-limiting examples of chemoattractants include IL-8, GCP-2,GRO-α, GRO-β, MGSA-β, MGSA-γ, PF₄,ENA-78, GCP-2, NAP-2, IL-8, IP10,I-309, I-TAC, SDF-1, BLC, BRAK, bolekine, ELC, LKTN-1, SCM-1β, MIG,MCAF, LD7α, eotaxin,, IP-110, HCC-1, HCC-2, Lkn-1, HCC-4, LARC, LEC,DC-CK1, PARC, AMAC-1, MIP-2β, ELC, exodus-3, ARC, exodus-1, 6Ckine,exodus 2, STCP-1, MPIF-1, MPIF-2, Eotaxin-2, TECK, Eotaxin-3, ILC, ITAC,BCA-1, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MCP-1, MCP-2, MCP-3, MCP-4,MCP-5, RANTES, eotaxin-1, eotaxin-2, TARC, MDC, TECK, CTACK, SLC,lymphotactin, and fractalkine; and other cells. Further non-limitingexamples of chemorepellants include receptor agonists and other cells.

[0081] In order to perform a test, such as a chemotaxis and/orchemoinvasion assay utilizing a soluble test substance, the test device10 is first fabricated. A preferred embodiment of the method of makingthe device according to the present invention will now be described. Amaster that is the negative of top-plate 11 is fabricated by standardphotolithographic procedures. A predetermined material is spin coated orinjection molded onto the master. The predetermined material is thencured, peeled off the master to comprise top member 11 and placed ontosupport member 16.

[0082] Where the test device 10 is a chemotaxis, haptotaxis and/orchemoinvasion device, a rigid frame with the standard microtiterfootprint is preferably placed around the outer perimeter of top member11. In one embodiment, a gel matrix is poured into well region 13 a andallowed to flow into channel region 15 a. After the gel matrix sets,excess gel is removed from well regions 13 a and 14 a. In anotherembodiment, no gel matrix is added to channel region 15 a. Subsequently,a biological sample of cells is placed in well region 14 a and a testsubstance is placed in well region 13 a. In one embodiment, a lowconcentration of a test substance is placed in well region 14 a in orderto activate the cells and expedite the beginning of the assay.Alternatively, depending on the cells being studied and the soluble testsubstance being used, the soluble test substance may be introducedduring or after the cells have been placed in well region 14 a. Once thesoluble test substance has been introduced, by the process of diffusion,a solution concentration gradient of the test substance forms along thelongitudinal axis of channel region 15 a from well region 13 acontaining the test agent towards well region 14 a containing thebiological sample of cells. A secondary effect of this solution gradientis the formation of a physisorbed (immobilized) gradient. When thissolution gradient is established, some fraction of the solute of thetest substance may adsorb onto support member 16. This adsorbed layer oftest solute may also contribute to chemotaxis and chemoinvasion. Thebiological sample of cells may respond to this concentration gradientand migrate towards the higher concentration of the test substance,migrate away from the higher concentration of the test substance, orexhibit inhibited movement in response to the higher concentration ofthe test substance. It is through this chemotaxis in response to thegradient, that the chemotactic influence of the chemotactic substancecan be measured. Chemotaxis is assayed by measuring the distance thecells travel and the amount of time the cells take to reach apredetermined point in the channel region 15 a or the distance the cellstravel and the amount of time the cells take to reach a certain point inwell region 14 a (in the case of a chemorepellant that causes cells tomove away from the chemotactic substance).

[0083] Utilizing an alternative embodiment of device 10 containing analternative design of chamber 12, a solution concentration gradient isformed using a network of microfluidic channel regions. In thisembodiment as seen in FIG. 14, first well region well region 13 a ofchamber 12 has first wells, 20, 21, and 22, connected by a network ofmicrofluidic capillaries 23 to channel 15. In particular, first wellregion 13 a includes a plurality of first wells connected by a pluralityof capillaries 24 connected to respective ones of the plurality of firstwells and a plurality of subcapillaries 25 branched off such that eachof the plurality of subcapillaries is connected to each of the pluralityof capillaries at one end thereof and to channel 15 at another endthereof. Each first well, 20, 21, and 22 receives a differentconcentration of soluble test substance. After the three first wells,20, 21, and 22 are simultaneously infused with the three differentconcentrations of soluble test substance, the solution streams traveldown the network of channel regions, continuously splitting, mixing andrecombining. After several generations of branched subcapillaries, eachsubcapillary containing different proportions of soluble test substancesare merged into a single channel 15, forming a concentration gradientacross channel 15, perpendicular to the flow direction.

[0084] According to one embodiment of the present invention,biomolecules are immobilized onto support member 16, preferably on theportion of upper surface U constituting the bottom surface of channel 15and of well region 13 a in any one of the embodiments of the test deviceof the present invention, such as the embodiments shown in FIGS. 1A-14.The concentration of biomolecules increases or decreases along thelongitudinal axis of the device from the upper surface of support member16 constituting the bottom surface of well region 13 a towards the uppersurface U of support member 16 constituting the bottom surface of wellregion 14 a thus forming a surface gradient. After the test biomoleculesare immobilized on support member 16, the top member is placed ontosupport member 16 and a rigid frame with the standard microtiterfootprint is placed around the outer perimeter of top member 11 andcells are added to well region 14 a. In an alternative embodiment, afterthe test biomolecules are immobilized on support member 16 and the topmember is placed over support member 16, a gel matrix is added tochannel region 15 a. Cells are subsequently added to well region 14 a.The biological sample of cells potentially respond to the concentrationgradient of immobilized biomolecules and migrates towards the higherconcentrations of the test biomolecules, away from the higherconcentrations of the test biomolecules, or exhibit inhibited migrationin response to the higher concentrations of the test biomolecules. Thesurface gradient can increase linearly or as a squared, cubed, orlogarithmic function or in any surface profile that can be approximatedin steps up or down.

[0085] The test biomolecules can be attached to and form surfacegradients on the upper surface U of support member 16 by variousspecific or non-specific approaches known in the art as described in K.Efimenko and J. Genzer, “How to Prepare Tunable Planar MolecularChemical Gradient,” 13 Applied Materials, 2001, No. 20, October 16; U.S.Pat. No. 5,514, incorporated herein by reference. For example,microcontact printing techniques, or any other method known in the art,can be used to immobilize on upper surface U of support member 16 alayer of SAMs presenting hexadecanethiol. Support member 16 is thenexposed to high energy light through a photolithographic mask of thedesired gradient micropattern or a grayscale mask with continuousgradations from white to black. When the mask is removed, a surfacegradient of SAMs presenting hexandecanethiol remains. Support member 16is then immersed in a solution of ethylene glycol terminatedalkanethiol. The regions of support member 16 with SAMs presentinghexadecanethiol will rapidly adsorb biomolecules and the regions of thesupport member with SAMs presenting oligomers of the ethylene glycolgroup will resist adsorption of protein. Support member 16 is thenimmersed in a solution of the desired test biomolecules and thebiomolecules rapidly adsorb only to the regions of support member 16containing SAMs presenting hexadecanethiol creating a surface gradientof immobilized biomolecules.

[0086] In another embodiment, the test biomolecules are immobilized onthe support member 16 and a surface concentration gradient forms afterthe top member 11 has been placed over support member 16 in any one ofthe embodiments of the test device of the present invention, such as theembodiments shown in FIGS. 1A-14. In this embodiment, discreteconcentrations of solution containing test biomolecules areconsecutively placed in well region 14 a and allowed to adsorbnon-specifically to support member 16. For example, first, a 1milligram/milliliter (mg/ml) of solution can first be placed in wellregion 14 a; second, a 1 microgram/milliliter (μg/ml) solution can beplaced in well region 14 a; last, a 1 nanogram/milliliter (ng/ml)solution of test biomolecules can be placed in well region 14 a. Thediffering concentrations of test biomolecules in solution result indiffering amounts of adsorption on support member 16.

[0087] Utilizing an alternative embodiment of device 10 containing analternative design of chamber 12 as seen in FIG. 11, an immobilizedbiomolecular surface gradient is formed based on the concept of laminarflow of multiple parallel liquid streams, a method known in the art.Based on this concept, when two or more streams with low Reynoldsnumbers are joined into a single stream, also with a low Reynoldsnumber, the combined streams flow parallel to each other withoutturbulent mixing. According to one embodiment, a solution of chemotacticbiomolecules is placed in 17 a and 19 a and a protein solution is placedin 18 a. The solutions are allowed to flow into channel region 15 aunder the influence of gentle aspiration at well region 14 a.Biomolecules adsorb nonspecifically to the regions of the surface overwhich the solution containing the biomolecules flows forming a surfacegradient. The wells are then filled with a suspension of cells andpotential haptotaxis of the cells towards the increasing concentrationgradient of biomolecules is observed and monitored. See generally, S.Takayama et al., “Patterning Cells and their Environment Using MultipleLaminar Fluid Flows in Capillary Networks” Pro. Natl. Acad. Sci. USA,Vol. 96, pp. 5545-5548, May 1999.

[0088] The present invention also contemplates an assay using both asoluble and surface gradient to determine whether the soluble testsubstance or the immobilized test biomolecules more heavily influencechemotaxis and chemoinvasion. In this embodiment, an assay is performedby forming a surface gradient as described above, an assay is performedby forming a solution gradient as described above, an assay is performedby forming both types of gradients and the results of all three assaysare compared. With respect to the combined gradient assay, testbiomolecules are immobilized on the upper surface U of support member 16constituting the bottom surface of well region 13 a and on the uppersurface of support member 16 underlying channel region 15 a and theconcentration of biomolecules decreases along the longitudinal axis ofchamber 12 from well region 13 a to well region 14 a, in any one of theembodiments of the test device of the present invention, such as theembodiments shown in FIGS. 1A-14. Additionally, a soluble test substanceis added to well region 13 a. Such an embodiment creates surface andsoluble chemotactic concentration gradients that decrease in the samedirection. If the combined concentration gradients have a synergisticeffect on chemotaxis and/or chemoinvasion, then both gradients should beused in screening both the cell receptor binding the chemotactic ligandsof the soluble chemotactic substance and the cell receptor binding theimmobilized biomolecules. Both types of receptors are identified asimportant and therapeutic agents that target both these receptors or acombination of therapeutic agents, one targeting one receptor andanother targeting the other receptor can be screened. If the combinedconcentration gradients do not have a synergistic effect, then theindividual gradient that more strongly promotes chemotaxis and/orchemoinvasion can be identified and the cell receptor that binds to thechemotactic ligands of the test agent forming the gradient can betargeted.

[0089] Identifying optimal chemotactic ligand and receptor pairs isimportant in understanding the biological pathways implicated inchemotaxis and/or chemoinvasion and developing therapeutic agents thattarget these pathways. Accordingly, the present invention generallyprovides using chemotactic test agents to determine which chemotacticreceptors expressed on a cell's surface most heavily influencechemotaxis and/or chemoinvasion. In one embodiment, the presentinvention provides for high throughput screening of a class ofchemoattractants known to attract a particular cell type having areceptor on the cell's surface for each chemoattractant within thisclass in order to identify which receptor is more strongly implicated inthe chemotaxis and/or chemoinvasion process. After identifying thisreceptor, the present invention contemplates high-throughput screeningof therapeutic agents that potentially block this receptor or bind tothis receptor, depending on whether chemotaxis and/or chemoinvasion isdesired to be promoted or prevented. In another embodiment, the presentinvention provides for high throughput screening of differentchemoattractants known to bind to the same receptor on a particular celltype's surface, in order to determine which chemoattractantligand/receptor pair more heavily influences chemotaxis and/orchemoinvasion. After identifying this ligand/receptor pair, the presentinvention contemplates high throughput screening of therapeutic agentsthat target this receptor and either block or activate this receptordepending one whether chemotaxis and/or chemoinvasion is desired to bepromoted or prevented.

[0090] The present invention also contemplates high-throughput screeningof a class of chemotactic inhibitors known to inhibit chemotaxis of aparticular cell type having various chemotactic receptors on the cell'ssurface in order identify which receptor is more strongly implicated inthe chemotaxis and chemoinvasion process. After identifying thisreceptor, the present invention provides for high throughput screeningof therapeutic agents that potentially block this receptor as well (ifsuch action is desired).

[0091] In one embodiment of the present invention, an assay is performedto determine whether a test compound inhibits cancer cell invasion. Inthis embodiment, untreated cancer cells are placed in well region 14 aand a test agent is placed in well region 13 a of chamber 12 in any oneof the embodiments of the test device of the present invention, such asthe embodiments shown in FIGS. 1A-14. Cell chemotaxis and invasion ismeasured and recorded. After a suitable test agent is identified (onethat chemically attracts the cancer cells) another assay is run inchamber 12. In this subsequent assay, cancer cells are placed in wellregion 14 a and a test compound, for example, a therapeutic, is alsoplaced in well region 14 a. In another embodiment, the test compound isalso placed in channel region 15 a. If a gel matrix is to be added tochannel region 15 a, the test compound can be mixed with the gel matrixbefore the gel is contacted with channel region 15 a during fabricationof device 10. A subsequent sample of the test agent identified in thefirst assay is placed in well region 13 a and the chemotaxis andinvasion of the cells treated with the test compound is compared to thechemotaxis and invasion of the cells not treated with the test compound.The test compound's anti-cancer potential is measured by whether thetreated cancer cells have a slower chemotaxis and invasion rate than theuntreated cancer cells.

[0092] With respect to another exemplary use of the chemotaxis andchemoinvasion device of the present invention, the device can be used toassay cells' response to the inflammatory response. A local infection orinjury in any tissue of the body attracts leukocytes into the damagedtissue as part of the inflammatory response. The inflammatory responseis mediated by a variety of signaling molecules produced within thedamaged tissue site by mast cells, platelets, nerve endings andleukocytes. Some of these mediators act on capillary endothelial cells,causing them to loosen their attachments to their neighboringendothelial cells so that the capillary becomes more permeable. Theendothelial cells are also stimulated to express cell-surface moleculesthat recognize specific carbohydrates that are present on the surface ofleukocytes in the blood and cause these leukocytes to adhere to theendothelial cells. Other mediators released from the damaged tissue actas chemoattractants, causing the bound leukocytes to migrate between thecapillary endothelial cells into the damaged tissue. To study leukocytechemotaxis, in one embodiment, channel region 15 a is treated tosimulate conditions in a human blood capillary during the inflammatoryresponse. For example, the side walls of channel region 15 a are coatedwith endothelial cells expressing cell surface molecules such asselecting, for example as shown in FIG. 4B. Leukocytes are then added towell region 14 a and a known chemoattractant is added to well region 13a in any one of the embodiments of the test device of the presentinvention, such as the embodiments shown in FIGS. 1A-14. Other suitablecell types that can be added to well region 14 a are neutrophils,monocytes, T and B lymphocytes, macrophages or other cell types involvedin response to injury or inflammation. The leukocytes' chemotaxis acrosschannel region 15 a towards well region 13 a is observed. Depending onthe type of infection to be studied, different categories of leukocytescan be used. For example, in one embodiment studying cell chemotaxis inresponse to a bacterial infection, well region 14 a receivesneutrophils. In another embodiment studying cell chemotaxis in responseto a viral infection, well region 14 a receives T-cells. In anotherembodiment simulating the process of angiogenesis, it is known in theart that growth factors applied to the cornea induce the growth of newblood vessels from the rim of highly vascularized tissue surrounding thecornea towards the sparsely vascularized center of the cornea. Thereforein another exemplary assay utilizing the chemotaxis and chemoinvasiondevice, cells from corneal tissue are placed in well region 13 a andendothelial cells are placed in well region 14 a in any one of theembodiments of the test device of the present invention, such as theembodiments shown in FIGS. 1A-14. A growth factor is added to wellregion 13 a and chemotaxis of the endothelial cells is observed,measured and recorded. Alternatively, since angiogenesis is alsoimportant in tumor growth (in order to supply oxygen and nutrients tothe tumor mass), instead of adding growth factor to well region 13 a,cancer cells from corneal tissue that produce angiogenic factors such asvascular endothelial growth factor (VEGF) could be added to well region13 a and normal endothelial cells added to well region 14 a. In adifferent embodiment also related to the study of angiogenesis, mastcells, macrophages, and fat cells that release fibroblast growth factorduring tissue repair, inflammation, and tissue growth are placed in wellregion 13 a and endothelial cells are placed in well region 14 a. Sinceduring angiogenesis, a capillary sprout grows into surroundingconnective tissue, to further simulate conditions in vivo, channelregion 15 a can be filled with a gel matrix.

[0093] There are several variations and embodiments of theaforementioned assays. One embodiment involves the number of channelsconnecting well region 13 a and well region 14 a of chamber 12 of device10. In one embodiment, such as the ones shown in FIGS. 3A-3C, there aremultiple channels connecting well region 13 a to well region 14 a. Byusing multiple channels, multiple assays can be performed simultaneouslyusing one biological sample of cells. In such an embodiment, all assaysare performed under uniform and consistent conditions and thereforeprovide statistically more accurate results. For example, each assaybegins with exactly the same number of potentially migratory cells andexactly the same concentration of test agent. Once a concentrationgradient forms, each assay is exposed to the gradient for the sameperiod of time. These multiple channels also provide redundancy in caseof failure in the assay.

[0094] Another embodiment of the cell invasion and chemotaxis assay ofthe present invention involves the placement of cells in well region 14a of chamber 12 in any one of the embodiments of the test device of thepresent invention, such as the embodiments shown in FIGS. 1A-14. Thecells may be patterned in a specific array on the upper surface U ofsupport member 16 constituting the bottom surface of well region 14 a ormay simply be deposited in no specific pattern or arrangement in wellregion 14 a. If the cells are patterned in a specific array on the uppersurface of support member 16 constituting the bottom surface of wellregion 14 a, then preferably, during the fabrication of device 10, theupper surface of support member 16 constituting the bottom surface ofwell region 14 a is first patterned with cells and then top member 11 isplaced over support member 16. It is desirable to monitor cellularmovement from a predetermined “starting” position to accurately measurethe distance and time periods the cells travel. As such, in oneembodiment, the cells are immobilized or patterned upon the supportmember underlying the first well in such a manner that the cells'viability is maintained and their position is definable so thatchemotaxis and invasion may be observed. There are several techniquesknown in the art to immobilize and pattern the cells into discreetarrays onto the support member. A preferred technique is described incopending application Ser. No. 60/330,456. In one embodiment, a cellposition patterning member is used to pattern the cells into definableareas onto the upper surface U of support member 16 constituting thebottom surface of well region 14 a of top member 11. If, for example,top member 11 is fabricated in the footprint of a standard 96-wellmicrotiter plate such that wells 13 and 14 correspond to the size andshape of the macrowells of the microtiter plate (not shown), then thecell position pattern member has outlined areas which correspond to thesize and shape of wells 13 and 14 and therefore correspond to the sizeand shape of the macrowells of the microtiter plate. Each outlined areahas micro through holes through which the cells will be patterned. Inorder to pattern the cells, the cell position patterning memberiscontacted with support member 16 and the outlined areas of the cellposition patterning member are aligned with portion of upper surface Uof support member 16 that constitutes the bottom surface of well region14 a, and will ultimately correspond to well region 14 a once top member11 is contacted with support member 16. Cells are then deposited overthe cell position patterning member and filter through the micro throughholes of the patterning member onto the support member underlying theareas corresponding to through-holes corresponding to second wellregions 14 a of chambers 12. Top member 11 is then placed over supportmember 16 such that through-holes 14 a are placed over the area ofsupport member 16 in which the cells are patterned. These patterningsteps result in discrete arrays of cells in well region 14 a.

[0095] Preferably, the cell position patterning member comprises anelastomeric material such as PDMS. Using PDMS for the patterning memberprovides a substantially fluid-tight seal between the patterning memberand the support member. This substantially fluid-tight seal ispreferable between these two components because cells placed in thewells are less likely to infiltrate adjoining wells if such a sealexists between the patterning member and the support member. Thearrangement of the micro through holes of the patterning member may berectangular, hexagonal, or another array resulting in the cells beingpatterned in these respective shapes. The width of each micro-throughhole may be varied according to cell types and desired number of cellsto be patterned. For example, if the width of both cell and microthrough hole is 10 microns, only one cell will deposit through eachmicro through hole. Thus, in this example, if the width of micro throughhole is 100 microns up to approximately 100 cells may be deposited.

[0096] The present invention also contemplates the patterning of morethan one cell type on the upper surface of support member 16constituting the bottom surface of well region 14 a in any one of theembodiments of the test device of the present invention, such as theembodiments shown in FIGS. 1A-14. Since cells of one type in vivo rarelyexist in isolation and are instead in contact and communication withother cell types, it is desirable to have a system in which cells can beassayed in an environment more like that of the body. For example, sincecancer cells are never found in isolation, but rather surrounded bynormal cells, an assay designed to test the effect of a drug on cancercells would be more accurate if the cancer cells in the assay weresurrounded by normal cells. In testing an anti-cancer drug, cancer cellsmay be patterned on the upper surface of support member 16 constitutingthe bottom surface of well region 14 a in any given one of theembodiments of the test device of the present invention, such as theembodiments of FIGS. 1A-14, and then through a separate patterningprocedure, the cancer cells may be surrounded by stromal cells. Topattern two different cell types on the upper surface of support member16 constituting the bottom surface of well region 14 a, a micro cellposition patterning member, as described above, is contacted withsupport member 16 and the outlined areas of the cell position patterningmember are aligned with the portion of upper surface U of support member16 that constitutes the bottom surface of well region 14 a, and willultimately correspond to well region 14 a once top member 11 iscontacted with support member 16. Cells of a first type may then bedeposited over the cell position patterning member and filter throughthe micro through holes of the patterning member onto the portion of theupper surface U of support member 16 constituting the bottom surface ofwell region 14 a. The micro cell position patterning member may then beremoved from support member 16. A macro cell position patterning memberwith outlined areas that correspond to the size and shape of wells 13and 14 and may therefore correspond to the size and shape of themacrowells of a 96 well microtiter plate. The macro cell positionpatterning member has macro through holes. A macro through hole of themacro cell position patterning member encompasses an area larger thanthe surface area defined by a micro through hole of the micro cellposition patterning member, but smaller than the surface area defined bywell region 14 a of chamber 12. The macro cell position patterningmember may then be contacted with support member 16. Cells of a secondtype may then be deposited over the macro cell position patterningmember and filter through the macro through holes of the macro cellposition patterning member onto the portion of upper surface U ofsupport member 16 constituting the bottom surface of well regions 14 aonce top member 11 is contacted with support member 16. Such patterningarrangement may result in cells of a second type surrounding and“stacking” cells of a first type. If it is desired to only have thecells of the second type stack the cells of the first type, then thesame micro cell position patterning member used to deposit the firstcell type or a different micro cell position patterning member havingthe exact same configuration as the patterning member used to depositcells of a first type, may be used to deposit cells of a second type.After the cells are patterned on support member 16, top member 11 may becontacted with support member 16 such that through holes in top member11 corresponding to the well region 14 a encompass the areas patternedwith cells. This essentially results in cells being immobilized in aspecific array within well region 14 a.

[0097] Notwithstanding how many different cell types are patterned onthe upper surface of support member 16 constituting the bottom surfaceof well region 14 a, the cells may be patterned on the support memberthrough several methods known in the art. For example, the cells may bepatterned on support member 16 through the use of SAMS. There areseveral techniques known in the art to pattern cells through the use ofSAMs of which a few exemplary techniques disclosed in U.S. Pat. No.5,512,131 to Kumar et al., U.S. Pat. No. 5,620,850 to Bambad et al.,U.S. Pat. No. 5,721,131 to Rudolph et al., U.S. Pat. Nos. 5,776,748 and5,976,826 to Singhvi et al. are incorporated by reference herein.

[0098] Several methods are known in the art to tag the cells in order toobserve and measure the aforementioned parameters. In one embodiment, anunpurified sample containing a cell type of interest is incubated with astaining agent that is differentially absorbed by the various celltypes. The cells are then placed in well region 14 a of chamber 12 inany given one of the embodiments of the test device of the presentinvention, such as the embodiments of FIGS. 1A-14. Individual, stainedcells are then detected based upon color or intensity contrast, usingany suitable microscopy technique(s), and such cells are assignedpositional coordinates. In another embodiment, an unpurified cell sampleis incubated with one or more detectable reporters, each reportercapable of selectively binding to a specific cell type of interest andimparting a characteristic fluorescence to all labeled cells. The sampleis then placed in well region 14 a of chamber 12 in any given one of theembodiments of the test device of the present invention, such as theembodiments of FIGS. 1A-14. The sample is then irradiated with theappropriate wavelength light and fluorescing cells are detected andassigned positional coordinates. One skilled in the art will recognizethat a variety of methods for discriminating selected cells from othercomponents in an unpurified sample are available. For example, thesemethods can include dyes, radioisotopes, fluorescers, chemiluminescers,beads, enzymes, and antibodies. Specific labeling of cell types can beaccomplished, for example, utilizing fluorescently-labeled antibodies.The process of labeling cells is well known in the art as is the varietyof fluorescent dyes that may be used for labeling particular cell types.

[0099] Cells of a chosen type may be also differentiated in a mixed-cellpopulation, for example, using a detectable reporter or a selectedcombination of detectable reporters that selectively and/orpreferentially bind to such cells. Labeling may be accomplished, forexample, using monoclonal antibodies that bind selectively to expressedCDs, antigens, receptors, and the like. Examples of tumor cell antigensinclude CD13 and CD33 present on myeloid cells; CD10 and CD19 present onB-cells; and CD2, CD5, and CD7 present on T-cells. One of skill in theart will recognize that numerous markers are available that identifyvarious known cell markers. Moreover, additional markers are continuallybeing discovered. Any such markers, whether known now or discovered inthe future, that are useful in labeling cells may be exploited inpracticing the invention.

[0100] Since few, if any markers are absolutely specific to only asingle type of cell, it may be desirable to label at least two markers,each with a different label, for each chosen cell type. Detection ofmultiple labels for each chosen cell type should help to ensure that thechemotaxis and chemoinvasion analysis is limited only to the cells ofinterest.

[0101] The present invention further provides a test device comprising:support means; means mounted to the support means for defining adiscrete chamber with the support means by being placed in fluid-tight,conformal contact with the support means. The discrete chamber includesa first well region including at least one first well; a second wellregion including at least one second well, the second well regionfurther being horizontally offset with respect to the first well regionin a test orientation of the device; and a channel region including atleast one channel connecting the first well region and the second wellregion with one another. An example of the support means comprises thesupport member 16 shown in FIGS. 1A, 1B, 12 and 13, while an example ofthe means mounted to the support means comprises the top member 11 shownin FIGS. 1A-11, 13 and 14. Other such means would be well known bypersons skilled in the art.

[0102] From the foregoing, it will be observed that numerousmodifications and variations can be effected without departing from thetrue spirit and scope of the novel concept of the present invention. Forexample, different embodiments of a device of the present invention maybe combined. Embodiments of the present invention further contemplatedifferent types of assays, for example, an assay wherein the test agentcomprises a buffer solution instead of a chemotactic agent. In such anassay, cell migration through channel region 15 a in observed in theabsence of a chemotactic gradient.

[0103] It will be appreciated that the present disclosure is intended toset forth the exemplifications of the invention, and theexemplifications set forth are not intended to limit the invention tothe specific embodiments illustrated. The disclosure is intended tocover by the appended claims all such modifications as fall within thespirit and scope of the claims.

EXAMPLES Example 1 Procedure for Fabrication of Chemoinvasion Device

[0104] A silicon wafer (6 inches) is spin coated with photoresist(SU8-50) at 2000 rpm for 45 seconds. After baking the wafer on a hotplate at 115° C. for 10 minutes, the wafer is allowed to cool to roomtemperature. A mask aligner (EVG620) is used to expose the photoresistfilm through a photomask. Exposure of 45 seconds is followed by anotherhard bake at 115° C. for 10 minutes. The silicon wafer is allowed tocool to room temperature for over 30 minutes. The uncrosslinkedphotoresist is removed using propylene glycol methyl ether acetate(PGMEA). The wafer is dried under a stream of nitrogen, and thepatterned photoresist is ready for subsequent processing.

[0105] In one embodiment, the patterned photoresist is spin-coated withanother layer of SU8-100 at 1500 rpm for 45 seconds. A mask aligner isused to selectively expose macrofeatures (i.e. wells) of the top memberbut not expose channel regions connecting the wells and other areas ofthe top member. After post exposure processing and photoresist removal,the master contains multiple layered features. This step may be repeatedto introduce macro-features on the master, which have the height ofapproximately 3 mm.

[0106] When a PDMS prepolymer is cast against the master, it faithfullyreplicates the features in the master. When casting, PDMS is added in anamount slightly lower than the height of the macrofeatures. After curingthe PDMS for four hours at 65 degrees C., the PDMS is peeled off thesilicon master and thoroughly cleaned with soap and water and rinsedwith 100% ethanol. A glass support member is also cleaned and rinsedwith ethanol. The PDMS membrane and glass support member are plasmaoxidized for 1 minute with the sides that would be bonded togetherfacing upward. The PDMS membrane is then placed onto the glass supportmember and pressure is applied to remove any air bubbles that may haveformed between the PDMS membrane and the glass support member. Theassembled device is then cooled to 4° C. Within 15 minutes of the plasmaoxidation of the PDMS membrane and the glass support member, 20microliters (pt) of Matrigel (any other hydrogel may be used) is pouredinto the first well and allowed to flow into the capillaries. The deviceis placed at room temperature for 15 minutes to set the Matrigel. Excessgel is then removed from the wells of the top member using a vacuum anda Pasteur pipette.

Example 2 Cell Chemoinvasion Assay

[0107] Placement of Cells and Test Agent in Chamber

[0108] The first and second wells of a chamber of a top member arefilled with phosphate buffered saline solution, PBS. The bottom of thesecond well may be treated with fibronectin (1 mg/ml) or otherextracellular matrix protein for 30 minutes, followed by washing twicewith PBS. After aspirating PBS, astrocytoma cells (U87-MG) are plated in50 μl of freshly warmed medium in the second well (25,000 cells per wellof a 24-well plate, in volume of 50 ul of solution per well). The cellsdeposit through the second well of the chamber, and attach to the bottomof the second well.

[0109] Cells are left to attach and spread in the second well overnightin a 37° C. incubator. At the start of the experiment, the cell mediumis exchanged for fresh serum-free medium. 10 μg of bFGF (basicfibroblast growth factor) per ml of medium is added to the first well ofeach chamber.

[0110] Image Acquisition and Data Analysis

[0111] Digital Images are taken on a Zeiss inverted microscope usingAXIOCAM™. Data was analyzed on AXIOVISION™ software. Time-lapsed imagesare taken every day at the same time for four days.

Example 3 Cell chemoinvasion Inhibition Assay Using Solution Gradient

[0112] Placement of Cells and Test Agent in Chambers

[0113] With respect to three chambers, the wells of each chamber of atop member are filled with PBS. The bottom of the second wells may betreated with fibronectin (1 mg/ml) or other extracellular matrix proteinfor 30 minutes, followed by washing twice with PBS. After aspiratingPBS, U87-MG cells are plated in 50 μl of freshly warmed medium in thesecond wells (10,000 cells per well of a 24-well plate, in volume of 50μl of medium per well). The cells deposit through the second wells ofeach chamber, and adhere to the bottom of the second wells.

[0114] Cells are left to attach and spread in the second wells overnightin a 37° C. incubator. At the start of the experiment, the cell mediumis exchanged for fresh serum-free medium or 1% serum. 1 μg of bFGF(basic fibroblast growth factor) per ml of medium is added to the firstwells of the chamber. A solution gradient is allowed to form for onehour.

[0115] With respect to the three different chambers, 100 μM of LY294002are placed in the second well of chamber #1, 10 μM LY294002 of areplaced in the second well of chamber #2, and 1.0 μM of LY294002 areplaced in the second well of chamber #3.

[0116] Image Acquisition and Data Analysis

[0117] Digital Images are taken on a Zeiss inverted microscope usingAXIOCAM™. Data was analyzed on AXIOVISION™ software. Time-lapsed imagesare taken every day at the same time for four days.

Example 4 Immobilization of Biomolecules on Support Member

[0118] After assembling the device as described above, the channelregions are filled with ethanolic solution containing (CH₃CH₂O)₃Si(CH₂)₃NH₂. After 20 minutes at room temperature, the channel regions arewashed off using ethanol. The device is incubated at 105° C. for onehour to crosslink the siloxane monolayer formed on the support member.The device is washed with ethanol to remove residues. The channelregions are filled with a solution of diisocyanate, either hexamethylenediisocyanate or tolyl diisocyanate (1% in acetonitrile or N-methylpyrrolidinone). The diisocyanate is allowed to react for two hours withthe terminal amino groups of the siloxane monolayer formed on thesupport member. The diisocyanate is washed off. The channel regions arefilled with 1 mg/ml solution of heparan sulfate or other sulfatedcarbohydrates (for example, di-acetylated form of heparin, heparinfragments, lectins containing sulfated sugars, etc.) The heparan sulfateis allowed to react with the support member to form immobilized species.The heparan sulfate solution and other reagents are washed off. Achemokine solution (any chemokine from CC, CXC, CX3C, or XC families maybe used) is introduced into the channel region. By electrostaticinteraction, chemokines that have higher pI (˜9-10) adsorb onto thenegatively charged sulfated support member.

Example 5 Chemotaxis Inhibition Assay Using Surface Gradient

[0119] Two wells are filled with 50 μl of PBS, and hydrostatic pressureis allowed to equalize. 5 μl of anti-hisx6 antibody are added to thefirst well and 5 μl of buffer are added to the second well to equalizehydrostatic pressure. By diffusion, the antibody concentration forms agradient from the first well to the second well. After 2 hours at roomtemperature, the two wells are washed off by adding 50 μl of buffer tothe second well and removing 50 μl from the first well. Byphysisorption, the solution gradient is transferred onto a surfacethereby forming a surface gradient. A solution of IL-8 (recombinanthuman IL-8 with a HISx6 fusion tag, R+D systems, catalog No. 968-IL) atconcentration of 25 μg/ml is added to the channel regions. The solutionis allowed to incubate for 30 minutes at room temperature. Excess IL-8chemokine is washed off and the surface is decorated with bound IL-8.Neutrophils(freshly isolated from a healthy donor) are added to thesecond well. Typically 20,000-100,000 cells are added in volume rangingfrom 10-550 μl. Neutrophils are allowed to adhere to the support memberand allowed to migrate towards the higher concentration of IL-8.Inhibition of migration is achieved by adding polyclonal antibodyagainst IL-8.

What is claimed is:
 1. A method of monitoring haptotaxis comprising:providing a device for monitoring haptotaxis having a housing defining achamber, the chamber including: a first well region including at leastone first well, the first well region configured to receive a test agenttherein and further including biomolecules immobilized therein; a secondwell region including at least one second well, the second well regionconfigured to receive a sample comprising cells therein and furtherbeing horizontally offset with respect to the first well region in atest orientation of the device; and a channel region with biomoleculesimmobilized therein and including at least one channel connecting thefirst well region and the second well region with one another. forming asurface concentration gradient along a longitudinal axis of the chamberby decreasing the concentration of biomolecules from the at least onefirst well to the at least one second well; placing a first samplecomprising cells in the at least one second well; and monitoringhaptotaxis of the cells.
 2. The method of claim 1, wherein the discreteassay chamber comprises a plurality of discrete assay chambers.
 3. Themethod of claim 2, wherein each of the first well region and the secondwell region of the plurality of discrete assay chambers are disposedrelative to one another to match a pitch of a standard microtiter plate.4. The method of claim 2, wherein the plurality of chambers is disposedrelative ton one another to match a pitch of a standard microtiterplate.
 5. The method of claim 1, wherein the at least one channelcomprises a plurality of channels.
 6. The method of claim 1, wherein thetop member is in reversible, fluid tight conformal contact with thesupport member.
 7. The method of claim 1, wherein a soluble testsubstance is added to the at least one first well to form a solutionconcentration gradient along the longitudinal axis of the chamber. 8.The method of claim 7, wherein the soluble test substance is achemoattractant, chemorepellant, or chemotactic inhibitor.
 9. The methodof claim 1, wherein the biomolecules are chemoattractants,chemorepellants, or chemotactic inhibitors.
 10. The method of claim 1,wherein the biomolecules are chemokines, cytokines, or small molecules.11. The method of claim 1, further comprising adding a test compound tothe at least one second well.
 12. The method of claim 11, wherein thetest compound is a therapeutic agent, a chemotactic inhibitor, achemoattractant, or a chemorepellant.
 13. The method of claim 1, whereinthe biomolecules are immobilized in the first well region and thechannel region through the use of SAMs.
 14. The method of claim 1,wherein placing the first sample comprising cells in the at least secondwell comprises patterning the cells in discrete arrays.
 15. The methodof claim 1, further comprising placing a second sample comprising cellsin the at least one second well.